Project Summary/Abstract Alcohol drinking is very common in the United States, with a majority of Americans having consumed alcohol within the past month. Roughly 7% of these individuals engage in heavy drinking and develop alcohol use disorders (AUDs), which produce significant economic and medical burdens on society. As problem drinking is characterized by consumption of intoxicating doses exceeding 80mg/dl (17.4mM), the majority of alcohol research targets blood alcohol concentrations (BACs) above this level. However, the effect of low concentrations of alcohol (<10mM) evident in early stages of drinking has not been well characterized. Identification of the neurobiological consequences of low-dose alcohol exposure can lead to a better understanding of the mechanisms involved in the initial reinforcing effects of alcohol and may ultimately lead to the development of better prevention efforts in mitigating subsequent alcohol misuse. We have shown that self- administration of low doses of ethanol in rats can lead to significant reinstatement of ethanol seeking, a model of ?craving?-like behavior. These doses of ethanol have also been shown to cause brain activation in regions known to be involved in reward. Importantly, self-administered ethanol produces unique patterns of neuronal activation in these brain areas that is not evident when ethanol is delivered non-contingently. We therefore propose to characterize the molecular targets of experimenter- versus self-administered ethanol at low doses using a phosphoproteomic approach. In Aim 1, we will use discovery-based quantitative label free mass spectrometry to identify differentially phosphorylated proteins in targeted brain regions known to be involved in ethanol reinforcement (nucleus accumbens, central, and basolateral nuclei of the amygdala, etc.) in male and female rats receiving chronic non-contingent injections of low (3mM), moderate (17.4mM), and high doses of ethanol (50mM) consistent with self-administration, binge drinking, and excessive drinking, respectively. Rats will then be euthanized to measure protein phosphorylation events, which are the major drivers of change in the functional activity of proteins, as a function of ethanol concentration. Aim 2 will extend these findings in rats trained to self-administer ethanol vs. yoked controls to address whether expectancy shifts the response of low- dose ethanol in the brain. We predict that ethanol will produce dose-dependent alterations in protein phosphorylation in terms of both magnitude and pattern of brain regions that are affected. Similarly, as female rats self-administer greater quantities of ethanol than males, we also expect to find sex-dependent patterns of protein regulation. Finally, we predict that self-administered ethanol will produce a unique pattern of neuronal activation compared to a similar low dose of experimenter-administered ethanol that may confer sensitivity to subsequent ethanol-motivated behavior. Overall, these experiments will help identify novel targets of low-dose ethanol that can be explored in future studies aimed at minimizing the risk of developing alcohol use disorders.